With the development of genetic engineering over the last two decades, including restriction enzymes, reverse transcriptase, cloning, polymerase chain reaction, sequencing, and monoclonal antibodies, there has been an extraordinary increase in the ability to isolate, identify and manipulate nucleic acid sequences. As a result of these capabilities, numerous genes and their transcriptional control elements have been identified and manipulated. The genes have been used for producing large amounts of a desired protein in heterologous hosts (bacterial and eukaryotic host cell systems).
In many cases, the process of obtaining coding sequences and eliciting their expression has been a long and arduous one. The identification of the coding sequence, either cDNA or genomic DNA, has frequently involved the construction of libraries, identification of fragments of the open reading frame, examining the flanking sequence, and the like. In mammalian genes where introns are frequently encountered, in many instances, the coding region has been only a small fraction of the total nucleic acid associated with the gene. In other cases, pseudogenes or multi-membered gene families have obscured the ability to isolate a particular gene of interest. Nevertheless, as techniques have improved, there has been a continuous parade of successful identifications and isolation of genes of interest.
For many reasons, it may be desirable to manipulate the coding region or the transcriptional regulatory regions without isolating the coding region or cloning the coding region on a fragment where the coding region is the primary sequence. These reasons may includes ease of manipulation, development of different pathways for expression, or the like.
Also, in many situations, one is primarily interested in a source of the protein product. The cell type in the body which produces the product is frequently an inadequate source. There is, therefore, significant interest in developing alternative techniques for producing proteins of interest in culture, with cells which provide for economic and efficient production of the desired protein and, when possible, appropriate processing of the protein product.
Relevant Literature
Mansour et al., Nature, 336:348-352 (1988), describe a general strategy for targeting mutations to non-selectable genes. Weidle et al., Gene, 66:193-203, (1988), describe amplification of tissue-type plasminogen activator with a DHFR gene and loss of amplification in the absence of selective pressure. Murnane and Yezzi, Somatic Cell and Molecular Genetics, 14:273-286, (1988), describe transformation of a human cell line with an integrated selectable gene marker lacking a transcriptional promoter, with tandem duplication and amplification of the gene marker. Thomas and Capecchi, Cell, 51:503-512, (19871, describe site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Song et al., Proc. Natl. Acad. Sci. USA, 84:6820-6824, (1987), describe homologous recombination in human cells by a two staged integration. Liskay et al., "Homologous Recombination Between Repeated Chromosomal Sequences in Mouse Cells," Cold Spring Harbor, Symp. Quant. Biol. 49:13-189, (1984), describe integration of two different mutations of the same gene and homologous recombination between the mutant genes. Rubnitz and Subramani, Mol. and Cell. Biol. 4:2253-2258, (1984), describe the minimum amount of homology required for homologous recombination in mammalian cells. Kim and Smithies, Nucl. Acids. Res. 16:8887-8903, (1988), describe an assay for homologous recombination using the polymerase chain reaction.
Burke, et al., Science 236:806-812 (1987) describe yeast artificial chromosomes (YACs). See also, Garza, et al., Science 246:641-646 (1989) and Brownstein, et al. Science 244:1348-1351 (1989).
See also, U.S. application Ser. No. 432,069, filed Nov. 6, 1989 and Ser. Nos. 466,088, filed Jan. 12, 1990 and 610,515, filed Nov. 11, 1990, which applications are incorporated herein by reference.